Ultrasound contrast agents and methods of making and using them

ABSTRACT

Gas or air filled microbubble suspensions in aqueous phases usable as imaging contrast agents in ultrasonic echography. They contain laminarized surfactants and, optionally, hydrophilic stabilizers. The laminarized surfactants can be in the form of liposomes. The suspensions are obtained by exposing the laminarized surfactants to air or a gas before or after admixing with an aqueous phase. One can impart outstanding resistance against collapse under pressure to these gas-filled microbubbles used as contrast agents in ultrasonic echography by using as fillers gases whose solubility in water, expressed in liter of gas by liter of water under standard conditions, divided by the square root of the molecular weight does not exceed 0.003.

[0001] The invention also comprises dry compositions which, uponadmixing with an aqueous carrier liquid, will generate the foregoingsterile suspension of microbubbles thereafter usable as contrast agentfor ultrasonic echography and other purposes. The present invention alsoconcerns stable dispersions or compositions of gas filled microvesiclesin aqueous carrier liquids. These dispersions are generally usable formost kinds of applications requiring gases homogeneously dispersed inliquids. One notable application for such dispersions is to be injectedinto living beings, for instance for ultrasonic echography and othermedical applications. The invention also concerns the methods for makingthe foregoing compositions including some materials involved in thepreparations, for instance pressure-resistant gas-filled microbubbles,microcapsules and microballoons.

BACKGROUND

[0002] It is well known that microbodies of air or a gas (defined hereas microvesicles). e.g., microbubbles or microballoons, suspended in aliquid are exceptionally efficient ultrasound reflectors for echography.In this disclosure, the term “microbubble” specifically designates airor gas globules in suspension in a liquid which generally results fromthe introduction therein of air or a gas in divided form, the liquidpreferably also containing surfactants or tensides to control thesurface properties thereof and the stability of the bubbles. Morespecifically, one may consider that the internal volume of themicrobubbles is limited by the gas/liquid interface, or in other words,the microbubbles are only bounded by a rather evanescent envelopeinvolving the molecules of the liquid and surfactant loosely bound atthe gas to liquid junction boundary.

[0003] The term “microcapsule” or “microballoon” designates preferablyair or gas bodies with a material boundary or envelope formed ofmolecules other than that of the liquid of suspension, e.g., a polymermembrane wall. Both microbubbles and microballoons are useful asultrasonic contrast agents. For instance, injecting into theblood-stream of living bodies suspensions of gas microbubbles ormicroballoons (in the range of 0.5 to 10 μm) in a carrier liquid willstrongly reinforce ultrasonic echography imaging, thus aiding in thevisualization of internal organs. Imaging of vessels and internal organscan strongly help in medical diagnosis, for instance for the detectionof cardiovascular and other diseases.

[0004] The formation of suspensions of microbubbles in an injectableliquid carrier suitable for echography can follow various routes, suchas by the release of a gas dissolved under pressure in this liquid, orby a chemical reaction generating gaseous products, or by admixing withthe liquid soluble or insoluble solids containing air or gas trapped oradsorbed therein. For instance in DE-A-3529195 (Max-Planck Gesell.),there is disclosed a technique for generating 0.5-50 μm bubbles in whichan aqueous emulsified mixture containing a water soluble polymer, an oiland mineral salts is forced back and forth, together with a small amountof air, from one syringe into another through a small opening. Here,mechanical forces are responsible for the formation of bubbles in theliquid.

[0005] M. W. Keller et al. (J. Ultrasound Med. 5 (1986), 439-8) havereported subjecting to ultrasonic cavitation under atmospheric pressuresolutions containing high concentrations of solutes such as dextrose,Renografin-76, Iopamidol (an X-ray contrast agent), and the like. Therethe air is driven into the solution by the energy of cavitation.

[0006] Other techniques rely on the shaking of a carrier liquid in whichair containing microparticles have been incorporated, said carrierliquid usually containing, as stabilizers, viscosity enhancing agents,e.g., water soluble polypeptides or carbohydrates and/or surfactants. Itis effectively admitted that the stability of the microbubbles againstdecay or escape to the atmosphere is controlled by the viscosity andsurface properties of the carrier liquid. The air or gas in themicroparticles can consist of inter-particle or intra-crystallineentrapped gas, as well as surface adsorbed gas, or gas produced byreactions with the carrier liquid, usually aqueous. All this is fullydescribed for instance in EP-A-0052575 (Ultra Med. Inc.) in which thereare used aggregates of 1-50 μm particles of carbohydrates (e.g.,galactose, maltose, sorbitol, gluconic acid, sucrose, glucose and thelike) in aqueous solutions of glycols or polyglycols, or other watersoluble polymers.

[0007] Also, in EP-A-0123235 and EP-A-0122624 (Schering, see alsoEP-A-0320433) use is made of air trapped in solids. For instance,EP-A-0122624 claims a liquid carrier contrast composition for ultrasonicechography containing microparticles of a solid surfactant, the latterbeing optionally combined with microparticles of a non-surfactant. Asexplained in this latter document, the formation of air bubbles in thesolution results from the release of the air adsorbed on the surface ofthe particles, or trapped within the particle lattice, or caught betweenindividual particles, this being so when the particles are agitated withthe liquid carrier.

[0008] EP-A-0131540 (Schering) also discloses the preparation ofmicrobubbles suspensions in which a stabilized injectable carrierliquid, e.g., a physiological aqueous solution of salt, or a solution ofa sugar like maltose, dextrose, lactose or galactose, without viscosityenhancer, is mixed with microparticles (in the 0.1 to 1 μm range) of thesame sugars containing entrapped air. In order that the suspension ofbubbles can develop within the liquid carrier, the foregoing documentsrecommend that both liquid and solid components be violently agitatedtogether under sterile conditions; the agitation of both componentstogether is performed for a few seconds and, once made, the suspensionmust then be used immediately, i.e., it should be injected within 5-10minutes for echographic measurements; this indicates that the bubbles inthe suspensions are not longlived and one practical problem with the useof microbubbles suspensions for injection is lack of stability withtime. The present invention fully remedies this drawback.

[0009] In an attempt to cure the evanescence problem, microballoons,i.e., microvesicles with a material wall, have been developed. As saidbefore, while the microbubbles only have an immaterial or evanescentenvelope, i.e., they are only surrounded by a wall of liquid whosesurface tension is being modified by the presence of a surfactant, themicroballoons or microcapsules have a tangible envelope made ofsubstantive material, e.g., a polymeric membrane with definitemechanical strength. In other terms, they are microvesicles of materialin which the air or gas is more or less tightly encapsulated.

[0010] In U.S. Pat. No. 4,466,442 (Schering), there is disclosed aseries of different techniques for producing suspensions of gasmicrobubbles in a liquid carrier liquid carrier using (a) a solution ofa tenside (surfactant) in a carrier liquid (aqueous) and (b) a solutionof a viscosity enhancer as stabilizer. For generating the bubbles, thetechniques used there include forcing at high velocity a mixture of (a),(b) and air through a small aperture; or injecting (a) into (b) shortlybefore use together with a physiologically acceptable gas; or adding anacid to (a) and a carbonate to (b), both components being mixed togetherjust before use and the acid reacting with the carbonate to generate CO₂bubbles; or adding an over-pressurized gas to a mixture of (a) and (b)under storage, said gas being released into microbubbles at the timewhen the mixture is used for injection.

[0011] The tensides used in component (a) of U.S. Pat. No. 4,466,442comprise lecithins; esters and ethers of fatty acids and fatty alcoholswith polyoxyethylene and polyoxyethylated polyols like sorbitol, glycolsand glycerol, cholesterol; and polyoxy-ethylene-polyoxypropylenepolymers. The viscosity raising and stabilizing compounds include forinstance mono- and polysaccharides (glucose, lactose, sucrose, dextran,sorbitol); polyols, e.g., glycerol, polyglycols; and polypeptides likeproteins, gelatin, oxypolygelatin, plasma protein and the like.

[0012] In a typical preferred example of this latter document,equivalent volumes of (a) a 0.5% by weight aqueous solution of Pluronic®F-68 (a polyoxypropylene-polyoxyethylene polymer) and (b) a 10% lactosesolution are vigorously shaken together under sterile conditions (closedvials) to provide a suspension of microbubbles ready for use as anultrasonic contrast agent and lasting for at least 2 minutes. About 50%of the bubbles had a size below 50 μm.

[0013] Although the achievements of the prior art have merit, theysuffer from several drawbacks which strongly limit their practical useby doctors and hospitals, namely their relatively short life-span (whichmakes test reproducibility difficult), relative low initial bubbleconcentration (the number of bubbles rarely exceeds 10⁴-10⁵ bubbles/mland the count decreases rapidly with time) and poor reproducibility ofthe initial bubble count from test to test (which also makes comparisonsdifficult). Also it is admitted that for efficiently imaging certainorgans, e.g., the left heart, bubbles smaller than 50 μm, preferably inthe range of 0.5-10 μm, are required; with larger bubbles, there arerisks of clots and consecutive emboly.

[0014] Furthermore, the compulsory presence of solid microparticles orhigh concentrations of electrolytes and other relatively inert solutesin the carrier liquid may be undesirable physiologically in some cases.Finally, the suspensions are totally unstable under storage and cannotbe marketed as such; hence great skill is required to prepare themicrobubbles at the right moment just before use.

[0015] Of course there exists stable suspensions of microcapsules, i.e.,microballoons with a solid, air-sealed rigid polymeric membrane whichperfectly resist for long storage periods in suspension, which have beendeveloped to remedy this shortcoming (see for instance K. J. Widder,EP-A-0324938); however the properties of microcapsules in which a gas isentrapped inside solid membrane vesicles essentially differ from that ofthe gas microbubbles of the present invention and belong to a differentkind of art; for instance while the gas microbubbles discussed here willsimply escape or dissolve in the blood-stream when the stabilizers inthe carrier liquid are excreted or metabolized, the solid polymermaterial forming the walls of the aforementioned micro-balloons musteventually be disposed of by the organism being tested which may imposea serious afterburden upon it. Also capsules with solid, non-elasticmembrane may break irreversibly under variations of pressure.

Stabilized Microbubble Compositions of the Invention

[0016] The compositions of the present invention fully remedy theaforementioned pitfalls.

[0017] The term “lamellar form” defining the condition of at least aportion of the surfactant or surfactants of the present compositionindicates that the surfactants, in strong contrast with themicroparticles of the prior art (for instance EP-A-0123235), are in theform of thin films involving one or more molecular layers (in laminateform). Converting film forming surfactants into lamellar form can easilybe done for instance by high pressure homogenization or by sonicationunder acoustical or ultrasonic frequencies. In this connection, itshould be pointed out that the existence of liposomes is a well knownand useful illustration of cases in which surfactants, more particularlylipids, are in lamellar form.

[0018] Liposome solutions are aqueous suspensions of microscopicvesicles, generally spherically shaped, which hold substancesencapsulated therein. These vesicles are usually formed of one or moreconcentrically arranged layers (lamellae) of amphipathic compounds,i.e., compounds having a lipophobic hydrophilic moiety and a lipophilichydrophobic moiety. See for instance “Liposome Methodology”, Ed. L. D.Leserman et al, Inserm 136, 2-8 (May 1982). Many surfactants ortensides, including lipids, particularly phospholipids, can belaminarized to correspond to this kind of structure. In this invention,one preferably uses the lipids commonly used for making liposomes, forinstance the lecithins and other tensides disclosed in more detailhereafter, but this does in no way preclude the use of other surfactants-provided they can be formed into layers or films.

[0019] It is important to note that no confusion should be made betweenthe microbubbles of this invention and the disclosure of Ryan (U.S. Pat.No. 4,900,540) reporting the use of air or gas filled liposomes forechography. In this method Ryan encapsulates air or a gas withinliposomic vesicles; in embodiments of the present invention microbubblesof air or a gas are formed in a suspension of liposomes (i.e., liquidfilled liposomes) and the liposomes apparently stabilize themicrobubbles. In Ryan, the air is inside the liposomes, which means thatwithin the bounds of the presently used terminology, the air filledliposomes of Ryan belong to the class of microballoons and not to thatof the microbubbles.

[0020] Practically, to achieve the suspensions of microbubbles accordingto the invention, one may start with liposomes suspensions or solutionsprepared by any technique reported in the prior art, with the obviousdifference that in the present case the liposomic vesicles arepreferably “unloaded”, i.e., they do not need to keep encapsulatedtherein any foreign material other than the liquid of suspension as isnormally the object of classic liposomes. Hence, preferably, theliposomes of the present invention will contain an aqueous phaseidentical or similar to the aqueous phase of the solution itself. Thenair or a gas is introduced into the liposome solution so that asuspension of microbubbles will form, said suspension being stabilizedby the presence of the surfactants in lamellar form. Notwithstanding,the material making the liposome walls can be modified within the scopeof the present invention, for instance by covalently grafting thereonforeign molecules designed for specific purposes as will be explainedlater.

[0021] The preparation of liposome solutions has been abundantlydiscussed in many publications, e.g., U.S. Pat. No. 4,224,179 andWO-A-88/09165 and all citations mentioned therein. This prior art isused here as reference for exemplifying the various methods suitable forconverting film forming tensides into lamellar form. Another basicreference by M. C. Woodle and D. Papahadjopoulos is found in “Methods inEnzymology” 171 (1989), 193.

[0022] For instance, in a method disclosed in D. A. Tyrrell et al,Biochimica & Biophysica Acta 457 (1976), 259-302, a mixture of a lipidand an aqueous liquid carrier is subjected to violent agitation andthereafter sonicated at acoustic or ultrasonic frequencies at room orelevated temperature. In the present invention, it has been found thatsonication without agitation is convenient. Also, an apparatus formaking liposomes, a high pressure homogenizer such as theMicrofluidizer®, which can be purchased from Microfluidics Corp.,Newton, Mass. 02164 USA, can be used advantageously. Large volumes ofliposome solutions can be prepared with this apparatus under pressureswhich can reach 600-1200 bar.

[0023] In another method, according to the teaching of GB-A-2,134,869(Squibb), microparticles (10 μm or less) of a hydrosoluble carrier solid(NaCl, sucrose, lactose and other carbohydrates) are coated with anamphipathic agent; the dissolution of the coated carrier in an aqueousphase will yield liposomic vesicles. In GB-A-2,135,647 insolubleparticles, e.g., glass or resin microbeads are coated by moistening in asolution of a lipid in an organic solvent followed by removal of thesolvent by evaporation. The lipid coated microbeads are thereaftercontacted with an aqueous carrier phase, whereby liposomic vesicles willform in that carrier phase.

[0024] The introduction of air or gas into a liposome solution in orderto form therein a suspension of microbubbles can be effected by usualmeans, inter alia by injection, that is, forcing said air or gas throughtiny orifices into the liposome solution, or simply dissolving the gasin the solution by applying pressure and thereafter suddenly releasingthe pressure. Another way is to agitate or sonicate the liposomesolution in the presence of air or an entrappable gas. Also one cangenerate the formation of a gas within the solution of liposomes itself,for instance by a gas releasing chemical reaction, e.g., decomposing adissolved carbonate or bicarbonate by acid. The same effect can beobtained by dissolving under pressure a low boiling liquid, for instancebutane, in the aqueous phase and thereafter allowing said liquid to boilby suddenly releasing the pressure.

[0025] Notwithstanding, an advantageous method is to contact the drysurfactant in lamellar or thin film, form with air or an adsorbable orentrappable gas before introducing said surfactant into the liquidcarrier phase. In this regard, the method can be derived from thetechnique disclosed in GB-A-2,135,647, i.e., solid microparticles orbeads are dipped in a solution of a film forming surfactant (or mixtureof surfactants) in a volatile solvent, after which the solvent isevaporated and the beads are left in contact with air (or an adsorbablegas) for a time sufficient for that air to become superficially bound tothe surfactant layer. Thereafter, the beads coated with air filledsurfactant are put into a carrier liquid, usually water with or withoutadditives, whereby air bubbles will develop within the liquid by gentlemixing, violent agitation being entirely unnecessary. Then the solidbeads can be separated, for instance by filtration, from the microbubblesuspension which is remarkably stable with time.

[0026] Needless to say that, instead of insoluble beads or spheres, onemay use as supporting particles water soluble materials like thatdisclosed in GB-A-2,134,869 (carbohydrates or hydrophilic polymers),whereby said supporting particles will eventually dissolve and finalseparation of a solid becomes unnecessary. Furthermore in this case, thematerial of the particles can be selected to eventually act asstabilizer or viscosity enhancer wherever desired.

[0027] In a variant of the method, one may also start with dehydratedliposomes, i.e., liposomes which have been prepared normally by means ofconventional techniques in the form of aqueous solutions and thereafterdehydrated by usual means. e.g., such as disclosed in U.S. Pat. No.4,229,360 also incorporated herein by reference. One of the methods fordehydrating liposomes recommended in this reference is freeze-drying(lyophilization), i.e., the liposome solution is frozen and dried byevaporation (sublimation) under reduced pressure. Prior to effectingfreeze-drying, a hydrophilic stabilizer compound is dissolved in thesolution, for instance a carbohydrate like lactose or sucrose or ahydrophilic polymer like dextran, starch, PVP, PVA and the like. This isuseful in the present invention since such hydrophilic compounds alsoaid in homogenizing the microbubbles size distribution and enhancestability under storage. Actually making very dilute aqueous solutions(0.1-10% by weight) of freeze-dried liposomes stabilized with, forinstance, a 5:1 to 10:1 weight ratio of lactose to lipid enables toproduce aqueous microbubbles suspensions counting 10⁸-10⁹microbubbles/ml (size distribution mainly 0.5-10 μm) which are stablefor at least a month (and probably much longer) without significantobservable change. And this is obtained by simple dissolution of theair-stored dried liposomes without shaking or any violent agitation.Furthermore, the freeze-drying technique under reduced pressure is veryuseful because it permits, after drying, to restore the pressure abovethe dried liposomes with any entrappable gas, i.e., nitrogen, CO₂,argon, methane, freon, etc., whereby after dissolution of the liposomesprocessed under such conditions suspensions of microbubbles containingthe above gases are obtained.

[0028] Microbubbles suspensions formed by applying gas pressure on adilute solution of laminated lipids in water (0.1-10% by weight) andthereafter suddenly releasing the pressure have an even higher bubbleconcentration, e.g., in the order of 10¹⁰-10¹¹ bubbles/ml. However, theaverage bubble size is somewhat above 10 μm, e.g., in the 10-50 μmrange. In this case, bubble size distribution can be narrowed bycentrifugation and layer decantation.

[0029] The tensides or surfactants which are convenient in thisinvention can be selected from all amphipathic compounds capable offorming stable films in the presence of water and gases. The preferredsurfactants which can be laminarized include the lecithins(phosphatidyl-choline) and other phospholipids. inter alia phosphatidicacid (PA), phosphatidylinositol, phosphatidylethanolamine (PE),phosphatidylserine (PS), phosphatidylglycerol (PG), cardiolipin (CL),sphingomyelins, the plasmogens, the cerebrosides, etc. Examples ofsuitable lipids are the phospholipids in general, for example, naturallecithins, such as egg lecithin or soya bean lecithin, or syntheticlecithins such as saturated synthetic lecithins, for example,dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine ordistearoylphosphatidylcholine or unsaturated synthetic lecithins, suchas dioleylphosphatidylcholine or dilinoleylphosphatidylcholine, with egglecithin or soya bean lecithin being preferred. Additives likecholesterol and other substances (see below) can be added to one or moreof the foregoing lipids in proportions ranging from zero to 50% byweight.

[0030] Such additives may include other surfactants that can be used inadmixture with the film forming surfactants and most of which arerecited in the prior art discussed in the introduction of thisspecification. For instance, one may cite free fatty acids, esters offatty acids with polyoxyalkylene compounds like polyoxypropylene glycoland polyoxyalkylene glycol; ethers of fatty alcohols withpolyoxyalkylene glycols; esters of fatty acids with polyoxyalklatedsorbitan; soaps; glycerol-polyalkylene stearate;glycerol-polyoxyethylene ricinoleate; homo- and copolymers ofpolyalkylene glycols; polyethoxylated soya-oil and castor oil as well ashydrogenated derivatives; ethers and esters of sucrose or othercarbohydrates with fatty acids, fatty alcohols, these being optionallypolyoxyalkylated; mono- di and triglycerides of saturated or unsaturatedfatty acids; glycerides of soya-oil and sucrose. The amount of thenon-film forming tensides or surfactants can be up to 50% by weight ofthe total amount of surfactants in the composition but is preferablybetween zero and 30%.

[0031] The total amount of surfactants relative to the aqueous carrierliquid is best in the range of 0.01 to 25% by weight but quantities inthe range 0.5-5% are advantageous because one always tries to keep theamount of active substances in an injectable solution as low aspossible, this being to minimize the introduction of foreign materialsinto living beings even when they are harmless and physiologicallycompatible.

[0032] Further optional additives to the surfactants include:

[0033] a) substances which are known to provide a negative charge onliposomes, for example, phosphatidic acid, phosphatidyl-glycerol ordicetyl phosphate;

[0034] b) substances known to provide a positive charge, for example,stearyl amine, or stearyl amine acetate;

[0035] c) substances known to affect the physical properties of thelipid films in a more desirable way; for example, capro-lactam and/orsterols such as cholesterol, ergosterol, phytosterol, sitosterol,sitosterol pyroglutamate, 7-dehydro-cholesterol or lanosterol, mayaffect lipid films rigidity;

[0036] d) substances known to have antioxidant properties to improve thechemical stability of the components in the suspensions, such astocopherol, propyl gallate, ascorbyl palmitate, or butylated hydroxytoluene.

[0037] The aqueous carrier in this invention is mostly water withpossibly small quantities of physiologically compatible liquids such asisopropanol, glycerol, hexanol and the like (see for instanceEP-A-052575). In general the amount of the organic hydrosoluble liquidswill not exceed 5-10% by weight.

[0038] The present composition may also contain dissolved or suspendedtherein hydrophilic compounds and polymers defined generally under thename of viscosity enhancers or stabilizers. Although the presence ofsuch compounds is not compulsory for ensuring stability to the air orgas bubbles with time in the present dispersions, they are advantageousto give some kind of “body” to the solutions. When desired, the upperconcentrations of such additives when totally innocuous can be veryhigh, for instance up to 80-90% by weight of solution with Iopamidol andother iodinated X-ray contrast agents. However, with the viscosityenhancers like for instance sugars, e.g., lactose, sucrose, maltose,galactose, glucose, etc. or hydrophilic polymers like starch, dextran,polyvinyl alcohol, polyvinyl-pyrrolidone, dextrin, xanthan or partlyhydrolyzed cellulose oligomers, as well as proteins and polypeptides,the concentrations are best between about 1 and 40% by weight, a rangeof about 5-20% being preferred.

[0039] Like in the prior art, the injectable compositions of thisinvention can also contain physiologically acceptable electrolytes; anexample is an isotonic solution of salt.

[0040] The present invention naturally also includes dry storablepulverulent blends which can generate the present microbubble containingdispersions upon simple admixing with water or an aqueous carrier phase.Preferably such dry blends or formulations will contain all solidingredients necessary to provide the desired microbubbles suspensionsupon the simple addition of water, i.e., principally the surfactants inlamellar form containing trapped or adsorbed therein the air or gasrequired for microbubble formation, and accessorily the other non-filmforming surfactants, the viscosity enhancers and stabilizers andpossibly other optional additives. As said before, the air or gasentrapment by the laminated surfactants occurs by simply exposing saidsurfactants to the air (or gas) at room or superatmospheric pressure fora time sufficient to cause said air or gas to become entrapped withinthe surfactant. This period of time can be very short, e.g., in theorder of a few seconds to a few minutes although over-exposure, i.e.,storage under air or under a gaseous atmosphere is in no way harmful.What is important is that air can well contact as much as possible ofthe available surface of the laminated surfactant, i.e., the drymaterial should preferably be in a “fluffy” light flowing condition.This is precisely this condition which results from the freeze-drying ofan aqueous solution of liposomes and hydrophilic agent as disclosed inU.S. Pat. No. 4,229,360.

[0041] In general, the weight ratio of surfactants to hydrophilicviscosity enhancer in the dry formulations will be in the order of0.1:10 to 10:1, the further optional ingredients, if any, being presentin a ratio not exceeding 50% relative to the total of surfactants plusviscosity enhancers.

[0042] The dry blend formulations of this invention can be prepared byvery simple methods. As seen before, one preferred method is to firstprepare an aqueous solution in which the film forming lipids arelaminarized, for instance by sonication, or using any conventionaltechnique commonly used in the liposome field, this solution alsocontaining the other desired additives, i.e., viscosity enhancers,non-film forming surfactants, electrolyte, etc., and thereafter freezedrying to a free flowable powder which is then stored in the presence ofair or an entrappable gas.

[0043] The dry blend can be kept for any period of time in the dry stateand sold as such. For putting it into use, i.e., for preparing a gas orair microbubble suspension for ultrasonic imaging, one simply dissolvesa known weight of the dry pulverulent formulation in a sterile aqueousphase, e.g., water or a physiologically acceptable medium. The amount ofpowder will depend on the desired concentration of bubbles in theinjectable product, a count of about 10⁸-10⁹ bubbles/ml being generallythat from making a 5-20% by weight solution of the powder in water. Butnaturally this figure is only indicative, the amount of bubbles beingessentially dependent on the amount of air or gas trapped duringmanufacture of the dry powder. The manufacturing steps being undercontrol, the dissolution of the dry formulations will providemicrobubble suspensions with well reproducible counts.

[0044] The resulting microbubble suspensions (bubble in the 0.5-10 μmrange) are extraordinarily stable with time, the count originallymeasured at start staying unchanged or only little changed for weeks andeven months; the only observable change is a kind of segregation, thelarger bubbles (around 10 μm) tending to rise faster than the smallones.

[0045] It has also been found that the microbubbles suspensions of thisinvention can be diluted with very little loss in the number ofmicrobubbles to be expected from dilution, i.e., even in the case ofhigh dilution ratios, e.g., 1/10² to 1/10⁴, the microbubble countreduction accurately matches with the dilution ratio. This indicatesthat the stability of the bubbles depends on the surfactant in lamellarform rather than on the presence of stabilizers or viscosity enhancerslike in the prior art. This property is advantageous in regard toimaging test reproducibility as the bubbles are not affected by dilutionwith blood upon injection into a patient.

[0046] Another advantage of the bubbles of this invention versus themicrobubbles of the prior art surrounded by a rigid but breakablemembrane which may irreversibly fracture under stress is that when thepresent suspensions are subject to sudden pressure changes, the presentbubbles will momentarily contract elastically and then resume theiroriginal shape when the pressure is released. This is important inclinical practice when the microbubbles are pumped through the heart andtherefore are exposed to alternating pressure pulses.

[0047] The reasons why the microbubbles in this invention are so stableare not clearly understood. Since to prevent bubble escape the buoyancyforces should equilibrate with the retaining forces due to friction,i.e., to viscosity, it is theorized that the bubbles are probablysurrounded by the laminated surfactant. Whether this laminar surfactantis in the form of a continuous or discontinuous membrane, or even asclosed spheres attached to the microbubbles, is for the moment unknownbut under investigation. However the lack of a detailed knowledge of thephenomena presently involved does not prelude full industrialoperability of the present invention.

[0048] The bubble suspensions of the present invention are also usefulin other medical/diagnostic applications where it is desirable to targetthe stabilized microbubbles to specific sites in the body followingtheir injection, for instance to thrombi present in blood vessels, toatherosclerotic lesions (plaques) in arteries, to tumor cells, as wellas for the diagnosis of altered surfaces of body cavities, e.g.,ulceration sites in the stomach or tumors of the bladder. For this, onecan bind monoclonal antibodies tailored by genetic engineering, antibodyfragments or polypeptides designed to mimic antibodies, bioadhesivepolymers, lectins and other site-recognizing molecules to the surfactantlayer stabilizing the microbubbles. Thus monoclonal antibodies can bebound to phospholipid bilayers by the method described by L. D.Leserman, P. Machy and J. Barbet (“Liposome Technology vol. III” p. 29ed. by G. Gregoriadis, CRC Press 1984). In another approach a palmitoylantibody is first synthesized and then incorporated in phospholipidbilayers following L. Huang, A. Huang and S. J. Kennel (“LiposomeTechnology vol. III” p. 51 ed. by G. Gregoriadis, CRC Press 1984).Alternatively, some of the phospholipids used in the present inventioncan be carefully selected in order to obtain preferential uptake inorgans or tissues or increased half-life in blood. Thus GM1gangliosides- or phosphatidylinositol-containing liposomes, preferablyin addition to cholesterol, will lead to increased, half-lifes in bloodafter intravenous administration in analogy with A. Gabizon, D.Papahadjopoulos, Proc. Natl Acad. Sci USA 85 (1988) 6949.

[0049] The gases in the microbubbles of the present invention caninclude, in addition to current innocuous physiologically acceptablegases like CO₂, nitrogen, N₂O, methane, butane, freon and mixturesthereof, radioactive gases such as ¹³³ Xe or ⁸¹ Kr are of particularinterest in nuclear medicine for blood circulation measurements, forlung scintigraphy etc.

[0050] Although in conjunction with suitable surfactants andstabilizers, the-gases used may include gases like sulfur hexafluoride,tetrafluoromethane, chlorotrifluoromethane, dichlorodifluoro-methane,bromotrifluoromethane, bromochlorodifluoromethane,dibromo-difluoromethane dichlorotetrafluoroethane,chloropentafluoroethane, hexafluoroethane, hexafluoropropylene,octafluoropropane, hexafluoro-butadiene, octafluoro-2-butene,octafluorocyclobutane, decafluorobutane, perfluorocyclopentane,dodecafluoropentane and more preferably sulfur hexafluoride and/oroctafluorocyclobutane, may be used. The media of the inventionpreferably contains a gas that includes one selected from sulfurhexafluoride, tetrafluoromethane, hexafluoroethane,hexafluoro-propylene, octafluoropropane, hexafluorobutadiene,octafluoro-2-butene, octafluorocyclobutane, decafluorobutane,perfluorocyclopentane, dodecafluoropentane and more preferably sulfurhexafluoride and/or octafluorocyclobutane.

[0051] The invention described up until this point can be furtherelucidated by the description of the following representative (but notlimiting) embodiments, numbered 1-27:

[0052] 1. A composition adapted for injection into the bloodstream andbody cavities of living beings, e.g., for the purpose of ultrasonicechography consisting of a suspension of air or gas microbubbles in aphysiologically acceptable aqueous carrier phase comprising from about0.01 to about 20% by weight of one or more dissolved or dispersedsurfactants, characterized in that at least one of the surfactants is afilm forming surfactant present in the composition at least partially inlamellar or laminar form.

[0053] 2. The composition of embodiment 1, characterized in that thelamellar surfactant is in the form of mono- or pluri-molecular membranelayers.

[0054] 3. The composition of embodiment 1, characterized in that thelamellar surfactant is in the form of liposome vesicles.

[0055] 4. The composition of embodiment 1, characterized in that itessentially consists of a liposome solution containing air or gasmicrobubbles developed therein.

[0056] 5. The composition of embodiment 4, characterized in that thesize of most of both liposomes and microbubbles is below 50 μm,preferably below 10 μm.

[0057] 6. The composition of embodiment 1, containing about 10⁸-10⁹bubbles of 0.5-10 μm size/ml, said concentration showing little orsubstantially no variability under storage for at least a month.

[0058] 7. The composition of embodiment 1, characterized in that thesurfactants are selected from phospholipids including the lecithins suchas phosphatidic acid, phosphatidylcholine, phosphatidylethanolamine,phosphatidylserine, phosphatidylglycerol, phosphatidylinositol,cardiolipin and sphyngomyelin.

[0059] 8. The composition of embodiment 7, characterized in furthercontaining substances affecting the properties of liposomes selectedform phosphatidylglycerol, dicetylphosphate, cholesterol, ergosterol,phytosterol, sitosterol, lanosterol, tocopterol, propyl gallate,ascorbyl palmitate and butylated hydroxytoluene.

[0060] 9. The composition of embodiment 1, further containing dissolvedviscosity enhancers or stabilizers selected from linear and cross-linkedpoly- and oligo-saccharides, sugars, hydrophilic polymers and iodinatedcompounds such as lopamidol in a weight ratio to the surfactantscomprised between about 1:5 to 100:1.

[0061] 10. The composition of embodiment 1, in which the surfactantscomprise up to 50% by weight of non-laminar surfactants selected fromfatty acids, esters, and ethers of fatty acids and alcohols with polyolssuch as polyalkylene glycols, polyalkylenated sugars and othercarbohydrates, and polyalkylenated glycerol.

[0062] 11. A method for the preparation of the suspensions of embodiment1, characterized by the following steps:

[0063] (a) selecting at least one film forming surfactant and convertingit into lamellar form;

[0064] (b) contacting the surfactant in lamellar form with air or anadsorbable or entrappable gas for a time sufficient for that air or gasto become bound by said surfactant; and

[0065] (c) admixing the surfactant in lamellar form with an aqueousliquid carrier, whereby a stable dispersion of air or gas microbubblesin said liquid carrier will result.

[0066] 12. The method of embodiment 11, in which step (c) is broughtabout before step (b), the latter being effected by introducingpressurized air or gas into the liquid carrier and thereafter releasingthe pressure.

[0067] 13. The method of embodiment 11, in which step (c) is broughtabout by gentle mixing of the components, no shaking being necessary,whereby the air or gas bound to the lamellar surfactant in step (b) willdevelop into a suspension of stable microbubbles.

[0068] 14. The method of embodiments 11 or 12, in which the liquidcarrier contains dissolved therein stabilizer compounds selected fromhydrosoluble proteins, polypeptides, sugars, poly- and oligo-saccharidesand hydrophilic polymers.

[0069] 15. The method of embodiment 11, in which the conversion of step(a) is effected by coating the surfactant onto particles of soluble orinsoluble materials; step (b) is effected by letting the coatedparticles stand for a while under air or a gas; and step (c) is effectedby admixing the coated particles with an aqueous liquid carrier.

[0070] 16. The method of embodiment 11, in which the conversion of step(a) is effected by sonicating or homogenizing under high pressure anaqueous solution of film forming lipids, this operation leading, atleast partly, to the formation of liposomes.

[0071] 17. The method of embodiment 16, in which step (b) is effected byfreeze-drying the liposome containing solution, the latter optionallycontaining hydrophilic stabilizers and contacting the resultingfreeze-dried product with air or gas for a period of time.

[0072] 18. The method of embodiments 16 and 17, in which the watersolution of film forming lipids also contains viscosity enhancers orstabilizers selected from hydrophilic polymers and carbohydrates inweight ratio relative to the lipids comprised between 1:5 and 100:1.

[0073] 19. A dry pulverulent formulation which, upon dissolution inwater, will form an aqueous suspension of microbubbles for ultrasonicechography, characterized in containing one or more film formingsurfactants in laminar form and hydrosoluble stabilizers.

[0074] 20. The dry formulation of embodiment 19, in which thesurfactants in laminar form are in the form of fine layers deposited onthe surface of soluble or insoluble solid particulate material.

[0075] 21. The dry formulation of embodiment 20, in which the insolublesolid particles are glass or polymer beads.

[0076] 22. The dry formulation of embodiment 20, in which the solubleparticles are made of hydrosoluble carbohydrates, polysaccharides,synthetic polymers, albumin, gelatin or lopamidol.

[0077] 23. The dry formulation of embodiment 19, which comprisesfreeze-dried liposomes.

[0078] 24. The use of the injectable composition of embodiment 1 forultrasonic echography.

[0079] 25. The use of the injectable composition of embodiments 1-10 fortransporting in the blood-stream or body cavities bubbles of foreigngases active therapeutically or diagnostically.

[0080] 26. The composition of embodiment 4, in which the surfactantcomprises, bound thereto, bioactive species designed for specifictargeting purposes, e.g., for immobilizing the bubbles in specificallydefined sites in the circulatory system, or in organs, or in tissues.

[0081] 27. The composition of embodiment 4, in which the surfactantcomprises, bound thereto, bioactive species selected from monoclonalantibodies, antibody fragments or polypeptides designed to mimicantibodies, bioadhesive polymers, lectins and other receptor recognizingmolecules.

[0082] The following Examples further illustrate the invention from apractical standpoint.

Echogenic Measurements

[0083] Echogenicity measurements were performed in a pulse - echosystem- made of a plexiglas specimen-holder (diameter 30 mm) and atransducer holder immersed in a constant temperature water bath, apulser-receiver (Accutron M3010S) with for the receiving part anexternal pre-amplifier with a fixed gain of 40 dB and an internalamplifier with adjustable gain from −40 to +40 dB. A 10 MHz low-passfilter was inserted in the receiving part to improve the signal to noiseratio. The A/D board in the IBM PC was a Sonotek STR 832. Measurementswere carried out at 2.25, 3.5, 5 and 7.5 MHz.

EXAMPLE 1

[0084] A liposome solution (50 mg lipids per ml) was prepared indistilled water by the REV method (see F. Szoka Jr. and D.Papahadjopoulos, Proc. Natl. Acad. Sci. USA 75 (1978) 4194) usinghydrogenated soya lecithin (NC 95 H, Nattermann Chemie, Koln, W.Germany) and dicetylphosphate in a molar ratio 9/1. This liposomepreparation was extruded at 65° C. (to calibrate the vesicle size)through a 1 μm polycarbonate filter (Nucleopore). Two ml of thissolution were admixed with 5 ml of a 75% iopamidol solution in water and0.4 ml of air and the mixture was forced back and forth through a twosyringe system as disclosed in DE-A-3529195, while maintainingcontinuously a slight over-pressure. This resulted in the formation of asuspension of microbubbles of air in the liquid (10⁵-10⁶ bubbles per ml,bubble size 1-20 μm as estimated by light microscopy) which was stablefor several hours at room temperature. This suspension gave a strongecho signal when tested by ultrasonic echography at 7.5, 5, 3.5 and 2.25MHz.

EXAMPLE 2

[0085] A distilled water solution (100 ml) containing by weight 2% ofhydrogenated soya lecithin and dicetylphosphate in a 9/1 molar ratio wassonicated for 15 min at 60°-65° C. with a Branson probe sonifier (Type250). After cooling, the solution was centrifuged for 15 min at 10,000 gand the supernatant was recovered and lactose added to make a 7.5% b.w.solution. The solution was placed in a tight container in which apressure of 4 bar of nitrogen was established for a few minutes whileshaking the container. Afterwards, the pressure was released suddenlywhereby a highly concentrated bubble suspension was obtained (10¹⁰-10¹¹lbubbles/ml). The size distribution of the bubbles was however wider thanin Example 1, i.e., from about 1 to 50 μm. The suspension was verystable but after a few days a segregation occurred in the standingphase, the larger bubbles tending to concentrate in the upper layers ofthe suspension.

EXAMPLE 3

[0086] Twenty g of glass beads (diameter about 1 mm) were immersed intoa solution of 100 mg of dipalmitoylphosphatidylcholine (Fluka A. G.Buchs) in 10 ml of chloroform. The beads were rotated under reducedpressure in a rotating evaporator until all CHCl³ had escaped. Then thebeads were further rotated under atmospheric pressure for a few minutesand 10 ml of distilled water were added. The beads were removed and asuspension of air microbubbles was obtained which was shown to containabout 10⁶ bubbles/mil after examination under the microscope. Theaverage size of the bubbles was about 3-5 μm. The suspension was stablefor several days at least.

EXAMPLE 4

[0087] A hydrogenated soya lecithin/dicetylphosphate suspension in waterwas laminarized using the REV technique as described in Example 1. Twoml of the liposome preparation were added to 8 ml of 15% maltosesolution in distilled water. The resulting solution was frozen at −30°C., then lyophilized under 0.1 Torr. Complete sublimation of the ice wasobtained in a few hours. Thereafter, air pressure was restored in theevacuated container so that the lyophilized powder became saturated withair in a few minutes.

[0088] The dry powder was then dissolved in 10 ml of sterile water undergentle mixing, whereby a microbubble suspension (10⁸-10⁹ microbubblesper ml, dynamic viscosity <20 mPa.s) was obtained. This suspensioncontaining mostly bubbles in the 1-5 μm range was stable for a very longperiod, as numerous bubbles could still be detected after 2 monthsstanding. This microbubble suspension gave a strong response inultrasonic echography. If in this example the solution is frozen byspraying in air at −30° to −70° C. to obtain a frozen snow instead of amonolithic block and the snow is then evaporated under vacuum, excellentresults are obtained.

EXAMPLE 5

[0089] Two ml samples of the liposome solution obtained as described inExample 4 were mixed with 10 ml of an 5% aqueous solution of gelatin(sample 5A), human albumin (sample 5B), dextran (sample 5C) andiopamidol (sample 5D). All samples were lyophilized. Afterlyophilization and introduction of air, the various samples were gentlymixed with 20 ml of sterile water. In all cases, the bubbleconcentration was above 10⁸ bubbles per ml and almost all bubbles werebelow 10 μm. The procedure of the foregoing Example was repeated with 9ml of the liposome preparation (450 mg of lipids) and only one ml of a5% human albumin solution. After lyophilization, exposure to air andaddition of sterile water (20 ml), the resulting solution contained2×10⁸ bubbles per ml, most of the them below 10 μm.

EXAMPLE 6

[0090] Lactose (500 mg), finely milled to a particle size of 1-3 μm, wasmoistened with a chloroform (5 ml) solution of 100 mg ofdimyristoylphosphatidylcholine/cholesterol/dipalmitoylphosphatidic acid(from Fluka) in a molar ratio of 4:1:1 and thereafter evaporated undervacuum in a rotating evaporator. The resulting free flowing white powderwas rotated a few minutes under nitrogen at normal pressure andthereafter dissolved in 20 ml of sterile water. A microbubble suspensionwas obtained with about 10⁵-10⁶ microbubbles per ml in the 1-10 μm sizerange as ascertained by observation under the microscope. In thisExample, the weight ratio of coated surfactant to water-soluble carrierwas 1:5. Excellent results (10⁷-10⁸ microbubbles/ml) are also obtainedwhen reducing this ratio to lower values, i.e., down to 1:20, which willactually increases the surfactant efficiency for the intake of air, thatis, this will decrease the weight of surfactant necessary for producingthe same bubble count.

EXAMPLE 7

[0091] An aqueous solution containing 2% of hydrogenated soya lecithinand 0.4% of Pluronic® F68 (a non ionic polyoxyethylenepolyoxypropylenecopolymer surfactant) was sonicated as described in Example 2. Aftercooling and centrifugation, 5 ml of this solution were added to 5 ml ofa 15% maltose solution in water. The resulting solution was frozen at−30° C. and evaporated under 0.1 Torr. Then air pressure was restored inthe vessel containing the dry powder. This was left to stand in air fora few seconds, after which it was used to make a 10% by weight aqueoussolution which showed under the microscope to be a suspension of verytiny bubbles (below 10 μm); the bubble concentration was in the range of10⁷ bubbles per ml. This preparation gave a very strong response inultrasonic echography at 2.25, 3.5, 5 and 7.5 MHz.

EXAMPLE 8

[0092] Two-dimensional echocardiography was performed in an experimentaldog following peripheral vein injection of 0.1-2 ml of the preparationobtained in Example 4. Opacification of the left heart with clearoutlining of the endocardium was observed, thereby confirming that themicrobubbles (or at least a significant part of them) were able to crossthe pulmonary capillary circulation.

EXAMPLE 9

[0093] A phospholipid/maltose lyophilized powder was prepared asdescribed in Example 4. However, at the end of the lyophilization step,a ¹³³Xe containing gas mixture was introduced in the evacuated containerinstead of air. A few minutes later, sterile water was introduced andafter gentle mixing a microbubble suspension containing ¹³³Xe in the gasphase was produced. This microbubble suspension was injected into livingbodies to undertake investigations requiring use of ¹³³Xe as tracer.Excellent results were obtained.

EXAMPLE 10 Comparative

[0094] In U.S. Pat. No. 4,900,540, Ryan et al disclose gas filledliposomes for ultrasonic investigations. According to the citation,liposomes are formed by conventional means but with the addition of agas or gas precursor in the aqueous composition forming the liposomecore (col. 2, lines 15-27).

[0095] Using a gas precursor (bicarbonate) is detailed in Examples 1 and2 of the reference. Using an aqueous carrier with an added gas forencapsulating the gas in the liposomes (not exemplified by Ryan et al)will require that the gas be in the form of very small bubbles, i.e., ofsize similar or smaller than the size of the liposome vesicles.

[0096] Aqueous media in which air can be entrapped in the form of verysmall bubbles (2.5-5 μm) are disclosed in M. W. Keller et al, J.Ultrasound Med. 5 (1986), 413-498.

[0097] A quantity of 126 mg of egg lecithin and 27 mg of cholesterolwere dissolved in 9 ml of chloroform in a 200 ml round bottom flask. Thesolution of lipids was evaporated to dryness on a Rotavapor whereby afilm of the lipids was formed on the walls of the flask. A 10 ml of a50% by weight aqueous dextrose solution was sonicated for 5 rainaccording to M. W. Keller et al (ibid) to generate air microbubblestherein and the sonicated solution was added to the flask containing thefilm of lipid, whereby hand agitation of the vessel resulted intohydration of the phospholipids and formation of multilamellar liposomeswithin the bubbles containing carrier liquid.

[0098] After standing for a while, the resulting liposome suspension wassubjected to centrifugation under 5000 g for 15 min to remove from thecarrier the air not entrapped in the vesicles. It was also expected thatduring centrifugation, the air filled liposomes would segregate to thesurface by buoyancy.

[0099] After centrifugation the tubes were examined and showed a bottomresidue consisting of agglomerated dextrose filled liposomes and a clearsupernatant liquid with substantially no bubbles left. The quantity ofair filled liposomes having risen by buoyancy was negligibly small andcould not be ascertained.

EXAMPLE 11 Comparative

[0100] An injectable contrast composition was prepared according to Ryan(U.S. Pat. No. 4,900,540, col. 3, Example 1). Egg lecithin (126 mg) andcholesterol (27 mg) were dissolved in 9 ml of diethylether. To thesolution were added 3 ml of 0.2 molar aqueous bicarbonate and theresulting two phase systems was sonicated until becoming homogeneous.The mixture was evaporated in a Rotavapor apparatus and 3 ml of 0.2molar aqueous bicarbonate were added.

[0101] A 1 ml portion of the liposome suspension was injected into thejugular vein of an experimental rabbit, the animal being under conditionfor heart ultrasonic imaging using an Acuson 128-XP5 ultrasonic imager(7.5 transducer probe for imaging the heart). The probe provided across-sectional image of the right and left ventricles (mid-papillarymuscle). After injection, a light and transient (a few seconds) increasein the outline of the right ventricle was observed. The effect washowever much inferior to the effect observed using the preparation ofExample 4. No improvement of the imaging of the left ventricle was notedwhich probably indicates that the CO₂ loaded liposomes did not pass thepulmonary capillaries barrier.

Further Methods of the Invention and Gases Used Therein

[0102] Despite the many progresses achieved regarding the stabilityunder storage of aqueous microbubble suspensions, this being either inthe precursor or final preparation stage, there still remained until nowthe problem of vesicle durability when the suspensions are exposed tooverpressure, e.g., pressure variations such as that occurring afterinjection in the blood stream of a patient and consecutive to heartpulses, particularly in the left ventricle. Actually, the presentinventors have observed that, for instance in anaesthetized rabbits, thepressure variations are not sufficient to substantially alter the bubblecount for a period of time after injection. In contrast, in dogs andhuman patients, typical microbubbles or microballoons filled with commongases such as air, methane or CO₂ will collapse completely in a matterof seconds after injection due to the blood pressure effect. It becamehence important to solve the problem and to increase the useful life ofsuspensions of microbubbles and membrane bounded microballoons underpressure in order to ensure that echographic measurements can beperformed in vivo safely and reproducibly.

[0103] It should be mentioned at this stage that another category ofechogenic image enhancing agents has been proposed which resistoverpressures as they consist of plain microspheres with a porousstructure, such porosity containing air or a gas. Such microspheres aredisclosed for instance in WO-A-91/12823 (Delta Biotechnology),EP-A-0327490 (Schering) and EP-A-0458079 (Hoechst). The drawback withthe plain porous microspheres is that the encapsulated gas-filled freespace is generally too small for good echogenic response and the sphereslack adequate elasticity. Hence the preference generally remains withthe hollow microvesicles and a solution to the collapsing problem wassearched.

[0104] This problem has now been solved by using gases or gas mixturesin conformity with the criteria outlined in the embodiments shown below.Briefly, it has been found that when the echogenic microvesicles aremade in the presence of a gas, respectively are filled at least in partwith a gas, having physical properties in conformity with the equationbelow, then the microvesicles remarkably resist pressure >60 Torr afterinjection for a time sufficient to obtain reproducible echographicmeasurements:${\frac{s_{gas}}{s_{air}} \times \frac{\left. \sqrt{}{Mw}_{air} \right.}{\left. \sqrt{}{Mw}_{gas} \right.}} \leqq 1$

[0105] In the foregoing equation, “s” designates the solubilities inwater expressed as the “Bunsen” coefficients, i.e., as volume of gasdissolved by unit volume of water under standard conditions (1 bar, 25°C.), and under partial pressure of the given gas of 1 atm (see the GasEncyclopaedia, Elsevier 1976). Since, under such conditions anddefinitions, the solubility of air is 0.0167, and the square root of itsaverage molecular weight (Mw) is 5.39, the above relation simplifies to:

s_(gas)/{square root}Mw_(gas)≦0.0031

[0106] In the Examples to be found hereafter there is disclosed thetesting of echogenic microbubbles and microballoons (see the Tables)filled with a number of different gases and mixtures thereof, and thecorresponding resistance thereof to pressure increases, both in vivo andin vitro. In the Tables, the water solubility factors have also beentaken from the aforecited Gas Encyclopaedia from “L'Air Liquide”,Elsevier Publisher (1976).

[0107] The microvesicles in aqueous suspension containing gasesaccording to the invention include most microbubbles and microballoonsdisclosed until now for use as contrast agents for echography. Thepreferred microballoons are those disclosed in EP-A-0324938,PCT/EP91/01706 and EP-A-0458745; the preferred microbubbles are those ofthe compositions disclosed herein (e.g., supra) and in PCT/EP91/00620;these microbubbles are advantageously formed from an aqueous liquid anda dry powder (microvesicle precursors) containing lamellarizedfreeze-dried phospholipids and stabilizers; the microbubbles aredeveloped by agitation of this powder in admixture with the aqueousliquid carrier. The microballoons of EP-A-0458745 have a resilientinterfacially precipitated polymer membrane of controlled porosity. Theyare generally obtained from emulsions into microdroplets of polymersolutions in aqueous liquids, the polymer being subsequently caused toprecipitate from its solution to form a fibrogenic membrane at thedroplet/liquid interface, which process leads to the initial formationof liquid-filled microvesicles, the liquid core thereof being eventuallysubstituted by a gas.

[0108] In order to carry out the method of the present invention, i.e.,to form or fill the microvesicles, whose suspensions in aqueous carriersconstitute the desired echogenic additives, with the gases according tothe foregoing relation, one can either use, as a first embodiment, a twostep route consisting of (1) making the microvesicles from appropriatestarting materials by any suitable conventional technique in thepresence of any suitable gas, and (2) replacing this gas originally used(first gas) for preparing the microvesicles with a new gas (second gas)according to the invention (gas exchange technique).

[0109] Otherwise, according to a second embodiment, one can directlyprepare the desired suspensions by suitable usual methods under anatmosphere of the new gas according to the invention.

[0110] If one uses the two-step route, the initial gas can be firstremoved from the vesicles (for instance by evacuation under suction) andthereafter replaced by bringing the second gas into contact with theevacuated product, or alternatively, the vesicles still containing thefirst gas can be contacted with the second gas under conditions wherethe second gas will displace the first gas from the- vesicles (gassubstitution). For instance, the vesicle suspensions, or preferablyprecursors thereof (precursors here may mean the materials themicrovesicle envelopes are made of, or the materials which, uponagitation with an aqueous carrier liquid, will generate or develop theformation of microbubbles in this liquid), can be exposed to reducedpressure to evacuate the gas to be removed and then the ambient pressureis restored with the desired gas for substitution. This step can berepeated once or more times to ensure complete replacement of theoriginal gas by the new one. This embodiment applies particularly wellto precursor preparations stored dry, e.g., dry powders which willregenerate or develop the bubbles of the echogenic additive uponadmixing with an amount of carrier liquid. Hence, in one preferred casewhere microbubbles are to be formed from an aqueous phase and drylaminarized phospholipids, e.g., powders of dehydrated lyophilizedliposomes plus stabilizers, which powders are to be subsequentlydispersed under agitation in a liquid aqueous carrier phase, it isadvantageous to store this dry powder under an atmosphere of a gasselected according to the invention. A preparation of such kind willkeep indefinitely in this state and can be used at any time fordiagnosis, provided it is dispersed into sterile water before injection.

[0111] Otherwise, and this is particularly so when the gas exchange isapplied to a suspension of microvesicles in a liquid carrier phase, thelatter is flushed with the second gas until the replacement (partial orcomplete) is sufficient for the desired purpose. Flushing can beeffected by bubbling from a gas pipe or, in some cases, by simplysweeping the surface of the liquid containing the vesicles under gentleagitation with a stream (continuous or discontinuous) of the new gas. Inthis case, the replacement gas can be added only once in the flaskcontaining the suspension and allowed to stand as such for a while, orit can be renewed one or more times in order to assure that the degreeof renewal (gas exchange) is more or less complete.

[0112] Alternatively, in a second embodiment as said before, one willeffect the full preparation of the suspension of the echogenic additivesstarting with the usual precursors thereof (starting materials), asrecited in the prior art and operating according to usual means of saidprior art, but in the presence of the desired gases or mixture of gasesaccording to the invention instead of that of the prior art whichusually recites gases such as air, nitrogen, CO₂ and the like.

[0113] It should be noted that in general the preparation mode involvingone first type of gas for preparing the microvesicles and, thereafter,substituting the original gas by a second kind of gas, the latter beingintended to confer different echogenic properties to said microvesicles,has the following advantage: As will be best seen from the results inthe Examples hereinafter, the nature of the gas used for making themicrovesicles, particularly the microballoons with a polymer envelope,has a definitive influence on the overall size (i.e., the average meandiameter) of said microvesicles; for instance, the size of microballoonsprepared under air with precisely set conditions can be accuratelycontrolled to fall within a desired range, e.g., the 1 to 10 μm rangesuitable for echographying the left and right heart ventricles. This notso easy with other gases, particularly the gases in conformity with therequirements of the present invention; hence, when one wishes to obtainmicrovesicles in a given size range but filled with gases the nature ofwhich would render the direct preparation impossible or very hard, onewill much advantageously rely on the two-steps preparation route, i.e.,one will first prepare the microvesicles with a gas allowing moreaccurate diameter and count control, and thereafter replace the firstgas by a second gas by gas exchange.

[0114] In the description of the Experimental part that follows(Examples), gas-filled microvesicles suspended in water or other aqueoussolutions have been subjected to pressures over that of ambient. It wasnoted that when the overpressure reached a certain value (which isgenerally typical for a set of microsphere parameters and workingconditions like temperature, compression rate, nature of carrier liquidand its content of dissolved gas (the relative importance of thisparameter will be detailed hereinafter), nature of gas filler, type ofechogenic material, etc.), the microvesicles started to collapse, thebubble count progressively decreasing with further increasing thepressure until a complete disappearance of the sound reflector effectoccurred. This phenomenon was better followed optically, (nephelometricmeasurements) since it is paralleled by a corresponding change inoptical density, i.e., the transparency of the medium increases as thebubble progressively collapse. For this, the aqueous suspension ofmicrovesicles (or an appropriate dilution thereof was placed in aspectrophotometric cell maintained at 25° C. (standard conditions) andthe absorbance was measured continuously at 600 or 700 nm, while apositive hydrostatic overpressure was applied and gradually increased.The pressure was generated by means of a peristaltic pump (Gilson'sMini-puls) feeding a variable height liquid column connected to thespectrophotometric cell, the latter being sealed leak-proof. Thepressure was measured with a mercury manometer calibrated in Torr. Thecompression rate with time was found to be linearly correlated with thepump's speed (rpm's). The absorbance in the foregoing range was found tobe proportional to the microvesicle concentration in the carrier liquid.

[0115] The invention will now be further described with reference toFIG. 1 which is a graph which relates the bubble concentration (bubblecount), expressed in terms of optical density in the aforementionedrange, and the pressure applied over the bubble suspension. The data forpreparing the graph are taken from the experiments reported in Example15.

[0116]FIG. 1 shows graphically that the change of absorbance versuspressure is represented by a sigmoid-shaped curve. Up to a certainpressure value, the curve is nearly flat which indicates that thebubbles are stable. Then, a relatively fast absorbance drop occurs,which indicates the existence of a relatively narrow critical regionwithin which any pressure increase has a rather dramatic effect on thebubble count. When all the microvesicles have disappeared, the curvelevels off again. A critical point on this curve was selected in themiddle between the higher and lower optical readings, i.e., intermediatebetween the “full”-bubble (OD max) and the “no”-bubble (OD min)measurements, this actually corresponding where about 50% of the bubblesinitially present have disappeared, i.e., where the optical densityreading is about half the initial reading, this being set, in the graph,relative to the height at which the transparency of the pressurizedsuspension is maximal (base line). This point which is also in thevicinity where the slope of the curve is maximal is defined as thecritical pressure PC. It was found that for a given gas, PC does notonly depend on the aforementioned parameters but also, and particularlyso, on the actual concentration of gas (or gases) already dissolved inthe carrier liquid: the higher the gas concentration, the higher thecritical pressure. In this connection, one can therefore increase theresistance to collapse under pressure of the microvesicles by making thecarrier phase saturated with a soluble gas, the latter being the same,or not, (i.e., a different gas) as the one that fills the vesicles. Asan example, air-filled microvesicles could be made very resistant tooverpressures (>120 Torr) by using, as a carrier liquid, a saturatedsolution of CO₂. Unfortunately, this finding is of limited value in thediagnostic field since once the contrast agent is injected to thebloodstream of patients (the gas content of which is of course outsidecontrol), it becomes diluted therein to such an extent that the effectof the gas originally dissolved in the injected sample becomesnegligible.

[0117] Another readily accessible parameter to reproducibly compare theperformance of various gases as microsphere fillers is the width of thepressure interval (ΔP) limited by the pressure values under which thebubble counts (as expressed by the optical densities) is equal to the75% and 25% of the original bubble count. Now, it has been surprisinglyfound that for gases where the pressure difference ΔP=P₂₅-P₇₅ exceeds avalue of about 25-30 Torr, the killing effect of the blood pressure onthe gas-filled microvesicles is minimized, i.e., the actual decrease inthe bubble count is sufficiently slow not to impair the significance,accuracy and reproducibility of echographic measurements.

[0118] It was found, in addition, that the values of PC and ΔP alsodepend on the rate of rising the pressure in the test experimentsillustrated by FIG. 1, i.e., in a certain interval of pressure increaserates (e.g., in the range of several tens to several hundreds ofTorr/min), the higher the rate, the larger the values for PC and ΔP. Forthis reason, the comparisons effected under standard temperatureconditions were also carried out at the constant increase rate of 100Torr/min. It should however be noted that this effect of the pressureincrease rate on the measure of the PC and ΔP values levels off for veryhigh rates; for instance the values measured under rates of severalhundreds of Torr/min are not significantly different from those measuredunder conditions ruled by heart beats.

[0119] Although the very reasons why certain gases obey theaforementioned properties, while others do not, have not been entirelyclarified, it would appear that some relation possibly exists in which,in addition to molecular weight and water solubility, dissolutionkinetics, and perhaps other parameters, are involved. However theseparameters need not be known to practice the present invention since gaseligibility can be easily determined according to the aforediscussedcriteria.

[0120] The gaseous species which particularly suit the invention are,for instance, halogenated hydrocarbons like the freons and stablefluorinated chalcogenides like SF₆, SeF₆ and the like. Although inconjunction with suitable surfactants and stabilizers, the gases usedmay include gases like sulfur hexafluoride, tetrafluoromethane,chlorotrifluoromethane, dichlorodifluoro-methane, bromotrifluoromethane,bromochlorodifluoromethane, dibromo-difluoromethanedichlorotetrafluoroethane, chloropentafluoroethane, hexafluoroethane,hexafluoropropylene, octafluoropropane, hexafluoro-butadiene,octafluoro-2-butene, octafluorocyclobutane, decafluorobutane,perfluorocyclopentane, dodecafluoropentane and more preferably sulfurhexafluoride and/or octafluorocyclobutane, may be used. The media of theinvention preferably contains a gas that includes one selected fromsulfur hexafluoride, tetrafluoromethane, hexafluoroethane,hexafluoro-propylene, octafluoropropane, hexafluorobutadiene,octafluoro-2-butene, octafluorocyclobutane, decafluorobutane,perfluorocyclopentane, dodecafluoropentane and more preferably sulfurhexafluoride and/or octafluorocyclobutane.

[0121] It has been mentioned above that the degree of gas saturation ofthe liquid used as carrier for the microvesicles according to theinvention has an importance on the vesicle stability under pressurevariations. Indeed, when the carrier liquid in which the microvesiclesare dispersed for making the echogenic suspensions of the invention issaturated at equilibrium with a gas, preferably the same gas with whichthe microvesicles are filled, the resistance of the microvesicles tocollapse under variations of pressure is markedly increased. Thus, whenthe product to be used as a contrast agent is sold dry to be mixed justbefore use with the carrier liquid (see for instance the productsdisclosed in PCT/EP91/00620 mentioned hereinbefore), it is quiteadvantageous to use, for the dispersion, a gas saturated aqueouscarrier. Alternatively, when marketing ready-to-use microvesiclesuspensions as contrast agents for echography, one will advantageouslyuse as the carrier liquid for the preparation a gas saturated aqueoussolution; in this case the storage life of the suspension will beconsiderably increased and the product may be kept substantiallyunchanged (no substantial bubble count variation) for extended periods,for instance several weeks to several months, and even over a year inspecial cases. Saturation of the liquid with a gas may be effected mosteasily by simply bubbling the gas into the liquid for a period of timeat room temperature.

[0122] The invention described herein can be further elucidated by thedescription of the following representative (but not limiting)embodiments, numbered 1-18:

[0123] 1. A method for imparting resistance against collapsing tocontrast agents for ultrasonic echography which consist of gas-filledmicrovesicles in suspension in aqueous liquid carrier phases, i.e.,either microbubbles bounded by an evanescent gas/liquid interfacialclosed surface, or microballoons bounded by a material envelope, saidcollapsing resulting, at least in part, from pressure increaseseffective, e.g., when the said suspensions are injected into the bloodstream of patients, said method comprising forming said microvesicles inthe presence of a gas, or if the microvesicles are already made fillingthem with this gas, which is a physiologically acceptable gas, or gasmixture, at least a fraction of which has a solubility in waterexpressed in liters of gas by liter of water under standard conditionsdivided by the square root of the molecular weight in daltons which doesnot exceed 0.003.

[0124] 2. The method of embodiment 1, which is carried out in two steps,in the first step the microvesicles or dry precursors thereof areinitially prepared under an atmosphere of a first gas, then in thesecond step at least a fraction of the first gas is substantiallysubstituted by a second gas, the latter being said physiologicallyacceptable gas.

[0125] 3. The method of embodiment 1, in which the physiologicallyacceptable gas used is selected from SF₆ or Freon® such as CF₄, CBrF₃,C₄F₈, CClF₃, CCl₂F₂, C₂F₆, C₃F₈, C₄F₆, C₅F₁₀,C₅F₁₂, C₂ClF₅, CBrClF₂,C₂Cl₂F₄, CBr₂F₂ and C₄F₁₀

[0126] 4. The method of embodiment 2, in which the gas used in the firststep is a kind that allows effective control of the average size andconcentration of the microvesicles in the carrier liquid, and thephysiologically acceptable gas added in the second step ensuresprolonged useful echogenic life to the suspension for in vivo ultrasonicimaging.

[0127] 5. The method of embodiment 1, in which the aqueous phasecarrying the microbubbles contains dissolved film-forming surfactants inlamellar or laminar form, said surfactants stabilizing the microbubblesboundary at the gas to liquid interface.

[0128] 6. The method of embodiment 5, in which said surfactants compriseone or more phospholipids.

[0129] 7. The method of embodiment 6, in which at least part of thephospholipids are in the form of liposomes.

[0130] 8. The method of embodiment 6, in which at least one of thephospholipids is a diacylphosphatidyl compound wherein the acyl group isa C₁₆ fatty acid residue or a higher homologue thereof.

[0131] 9. The method of embodiments 1 and 2, in which the microballoonmaterial envelope is made of an organic polymeric membrane.

[0132] 10. The method of embodiment 9, in which the polymers of themembrane are selected from polylactic or polyglycolic acid and theircopolymers, reticulated serum albumin, reticulated haemoglobin,polystyrene, and esters of polyglutamic and polyaspartic acids.

[0133] 11. The method of embodiment 1, in which the forming of themicrovesicles with said physiologically acceptable gas is effected byalternately subjecting dry precursors thereof to reduced pressure andrestoring the pressure with said gas, and finally dispersing theprecursors in a liquid carrier.

[0134] 12. The method of embodiment 1, in which the filling of themicroballoons with said physiologically acceptable gas is effected bysimply flushing the suspension with said gas under ambient pressure.

[0135] 13. The method of embodiment 1, which comprises making themicrovesicles by any standard method known in the art but operatingunder an atmosphere composed at least in part of said gas.

[0136] 14. Suspensions of gas filled microvesicles distributed in anaqueous carrier liquid to be used as contrast agents in ultrasonicechography, characterized in that the gas is physiologically acceptableand such that at least a portion thereof has a solubility in water,expressed in liter of gas by liter of water under standard conditions,divided by the square root of the molecular weight which does not exceed0.003.

[0137] 15. The aqueous suspensions of embodiment 14, characterized inthat the gas is such that the pressure difference ΔP between thosepressures which, when applied under standard conditions and at a rate ofabout 100 Torr/min to the suspension cause the collapsing of about 75%,respectively 25%, of the microvesicles initially present, is at least 25Torr.

[0138] 16. Aqueous suspensions according to embodiment 14, in which themicrovesicles are microbubbles filled with said physiologicallyacceptable gas suspended in an aqueous carrier liquid containingphospholipids whose fatty acid residues contain 16 carbons or more.

[0139] 17. Contrast agents for echography in precursor form consistingof a dry powder comprising lyophilized liposomes and stabilizers, thispowder being dispersible in aqueous liquid carriers to form echogenicsuspensions of gas-filled microbubbles, characterized in that it isstored under an atmosphere comprising a physiologically acceptable gaswhose solubility in water, expressed in liter of gas by liter of waterunder standard conditions, divided by the square root of the molecularweight does not exceed 0.003.

[0140] 18. The contrast agent precursors of embodiment 17, in which- theliposomes comprise phospholipids whose fatty acid residues have 16 ormore carbon atoms.

[0141] The following Examples further illustrate various aspects of theinvention.

EXAMPLE 12

[0142] Albumin microvesicles filled with air or various gases wereprepared as described in EP-A-0324938 using a 10 ml calibrated syringefilled with a 5% human serum albumin (HSA) obtained from the BloodTransfusion Service, Red-Cross Organization, Bern, Switzerland. Asonicator probe (Sonifier Model 250 from Branson Ultrasonic Corp, USA)was lowered into the solution down to the 4 ml mark of the syringe andsonication was effected for 25 sec (energy setting=8). Then thesonicator probe was raised above the solution level up to the 6 ml markand sonication was resumed under the pulse mode (cycle=0.3) for 40 sec.After standing overnight at 40° C., a top layer containing most of themicrovesicles had formed by buoyancy and the bottom layer containingunused albumin debris of denatured protein and other insolubles wasdiscarded. After resuspending the microvesicles in fresh albuminsolution the mixture was allowed to settle again at room temperature andthe upper layer was finally collected. When the foregoing sequences werecarried out under the ambient atmosphere, air filled microballoons wereobtained. For obtaining microballoons filled with other gases, thealbumin solution was first purged with a new gas, then the foregoingoperational sequences were effected under a stream of this gas flowingon the surface of the solution; then at the end of the operations, thesuspension was placed in a glass bottle which was extensively purgedwith the desired gas before sealing.

[0143] The various suspensions of microballoons filled with differentgases were diluted to 1:10 with distilled water saturated at equilibriumwith air, then they were placed in an optical cell as described aboveand the absorbance was recorded while increasing steadily the pressureover the suspension. During the measurements, the suspensionstemperature was kept at 25° C.

[0144] The results are shown in the Table 1 below and are expressed interms of the critical pressure PC values registered for a series ofgases defined by names or formulae, the characteristic parameters ofsuch gases, i.e., Mw and water solubility being given, as well as theoriginal bubble count and bubble average size (mean diameter in volume).TABLE 1 Bubble Bubble S Count size PC gas {square root} Sample Gas MwSolubility (10⁸/ml) (μm) (Torr) Mw AFre1 CF₄ 88 .0038 0.8 5.1 120  .0004AFre2 CBrF₃ 149  .0045 0.1 11.1 104  .0004 ASF1 SF₆ 146  .005 13.9 6.2150  .0004 ASF2 SF₆ 146  .005 2.0 7.9 140  .0004 AN1 N₂ 28 .0144 0.4 7.862 .0027 A14 Air 29 .0167 3.1 11.9 53 .0031 A18 Air 29 .0167 3.8 9.2 52— A19 Air 29 .0167 1.9 9.5 51 — AMe1 CH₄ 16 .032 0.25 8.2 34 .008  AKr1Kr 84 .059 0.02 9.2 86 .006  AX1 Xe 131  .108 0.06 17.2 65 .009  AX2 Xe131  .108 0.03 16.5 89 .009 

[0145] From the results of Table 1, it is seen that the criticalpressure PC increases for gases of lower solubility and higher molecularweight. It can therefore be expected that microvesicles filled with suchgases will provide more durable echogenic signals in vivo. It can alsobe seen that average bubble size generally increases with gassolubility.

EXAMPLE 13

[0146] Aliquots (1 ml) of some of the microballoon suspensions preparedin Example 12 were injected in the Jugular vein of experimental rabbitsin order to test echogenicity in vivo. Imaging of the left and rightheart ventricles was carried out in the grey scale mode using an Acuson128-XP5 echography apparatus and a 7.5 MHz transducer. The duration ofcontrast enhancement in the left ventricle was determined by recordingthe signal for a period of time. The results are gathered in Table 2below which also shows the PC of the gases used. TABLE 2 Duration ofSample (Gas) contrast (sec) PC (Torr) AMe1 (CH₄) zero  34 A14 (air)  10 53 A18 (air)  11  52 AX1 (Xe)  20  65 AX2 (Xe)  30  89 ASF2 (SF₆) >60140

[0147] From the above results, one can see the existence of a definitecorrelation between the critical pressure of the gases tried and thepersistence in time of the echogenic signal.

EXAMPLE 14

[0148] A suspension of echogenic air-filled galactose microparticles(Echovist® from Schering AG) was obtained by shaking for 5 sec 3 g ofthe solid microparticles in 8.5 ml of a 20% galactose solution. In otherpreparations, the air above a portion of Echovist® particles wasevacuated (0.2 Torr) and replaced by an SF₆ atmosphere, whereby, afteraddition of the 20% galactose solution, a suspension of microparticlescontaining associated sulfur hexafluoride was obtained. Aliquots (1 ml)of the suspensions were administered to experimental rabbits (byinjection in the jugular vein) and imaging of the heart was effected asdescribed in the previous example. In this case the echogenicmicroparticles do not transit through the lung capillaries, henceimaging is restricted to the right ventricle and the overall signalpersistence has no particular significance. The results of Table 3 belowshow the value of signal peak intensity a few seconds after injection.TABLE 3 Sample No Gas Signal peak (arbitrary units) Gal1 air 114 Gal2air 108 Gal3 SF₆ 131 Gal4 SF₆ 140

[0149] It can be seen that sulfur hexafluoride, an inert gas with lowwater solubility, provides echogenic suspensions which generateechogenic signals stronger than comparable suspensions filled with air.These results are particularly interesting in view of the teachings ofEP-A-0441468 and EP-A-0357163 (Schering) which disclose the use forechography purposes of microparticles, respectively, cavitate andclathrate compounds filled with various gases including SF₆; thesedocuments do not however report particular advantages of SF₆ over othermore common gases with regard to the echogenic response.

EXAMPLE 15

[0150] A series of echogenic suspensions of gas-filled microbubbles wereprepared by the general method set forth below:

[0151] One gram of a mixture of hydrogenated soya lecithin (fromNattermann Phospholipids GmbH, Germany) and dicetyl-phosphate (DCP), in9/1 molar ratio, was dissolved in 50 ml of chloroform, and the solutionwas placed in a 100 ml round flask and evaporated to dryness on aRotavapor apparatus. Then, 20 ml of distilled water were added and themixture was slowly agitated at 75° C. for an hour. This resulted in theformation of a suspension of multilamellar liposomes (MLV) which wasthereafter extruded at 75° C. through, successively, 3 μm and 0.8 μmpolycarbonate membranes (Nuclepore(D)). After cooling, 1 ml aliquots ofthe extruded suspension were diluted with 9 ml of a concentrated lactosesolution (83 g/l), and the diluted suspensions were frozen at −45° C.The frozen samples were thereafter freeze-dried under high vacuum to afree-flowing powder in a vessel which was ultimately filled with air ora gas taken from a selection of gases as indicated in Table 4 below. Thepowdery samples were then resuspended in 10 ml of water as the carrierliquid, this being effected under a stream of the same gas used to fillthe said vessels. Suspension was effected by vigorously shaking for 1min on a vortex mixer.

[0152] The various suspensions were diluted 1:20 with distilled waterequilibrated beforehand with air at 25° C. and the dilutions were thenpressure tested at 25° C. as disclosed in Example 12 by measuring theoptical density in a spectrophotometric cell which was subjected to aprogressively increasing hydrostatic pressure until all bubbles hadcollapsed. The results are collected in Table 4 below which, in additionto the critical pressure PC, gives also the ΔP values (see FIG. 1).TABLE 4 Bubble increment Sample Solubility Count PC Δ P No Gas Mw in H₂O(10⁸/ml) (Torr) (Torr) LFre1 CF₄ 88 .0038 1.2 97 35 LFre2 CBrF₃ 149 .0045 0.9 116  64 LSF1 SF₆ 146  .005 1.2 92 58 LFre3 C₄F₈ 200  .016 1.5136  145  L1 air 29 .0167 15.5 68 17 L2 air 29 .0167 11.2 63 17 LAr1 Ar40 .031 14.5 71 18 LKr1 Kr 84 .059 12.2 86 18 LXe1 Xe 131  .108 10.1 9223 LFre4 CHClF₂ 86 .78 — 83 25

[0153] The foregoing results clearly indicate that the highestresistance to pressure increases is provided by the most water-insolublegases. The behavior of the microbubbles is therefore similar to that ofthe microballoons in this regard. Also, the less water-soluble gaseswith the higher -molecular weights provide the flattestbubble-collapse/pressure curves (i.e., ΔP is the widest) which is alsoan important factor of echogenic response durability in vivo, asindicated hereinbefore.

EXAMPLE 16

[0154] Some of the microbubble suspensions of Example 15 were injectedto the jugular vein of experimental rabbits as indicated in Example 13and imaging of the left heart ventricle was effected as indicatedpreviously. The duration of the period for which a useful echogenicsignal was detected was recorded and the results are shown in Table 5below in which C₄F₈ indicates octafluorocyclobutane. TABLE 5 Sample NoType of gas Contrast duration (sec) L1 Air  38 L2 Air  29 LMe1 CH₄  47LKr1 Krypton  37 LFre1 CF₄ >120 LFre2 CBrF₃  92 LSF1 SF₆ >112 LFre3 C₄F₈>120

[0155] These results indicate that, again in the case of microbubbles,the gases according to the criteria of the present invention willprovide ultrasonic echo signal for a much longer period than most gasesused until now.

EXAMPLE 17

[0156] Suspensions of microbubbles were prepared using different gasesexactly as described in Example 15, but replacing the lecithinphospholipid ingredient by a mole equivalent ofdiarachidoylphosphatidylcholine (C₂₀ fatty acid residue) available fromAvanti Polar Lipids, Birmingham, Ala. USA. The phospholipid to DCP molarratio was still 9/1. Then the suspensions were pressure tested as inExample 15; the results, collected in Table 6A below, are to be comparedwith those of Table 4. TABLE 6A Bubble Sample Type of Mw of SolubilityCount PC increment No Gas Gas in water (10⁸/ml) (Torr) Δ P (Torr) LFre1CF₄ 88 .0038 3.4 251  124  LFre2 CBrF₃ 149  .0045 0.7 121  74 LSF1 SF₆146  .005 3.1 347  >150  LFre3 C₄F₈ 200  .016 1.7 >350  >200  L1 Air 29.0167 3.8 60 22 LBu1 Butane 58 .027 0.4 64 26 LAr1 Argon 40 .031 3.3 8447 LMe1 CH₄ 16 .032 3.0 51 19 LEt1 C₂H₆ 44 .034 1.4 61 26 LKr1 Kr 84.059 2.7 63 18 LXe1 Xe 131  .108 1.4 60 28 LFre4 CHClF₂ 86 .78 0.4 58 28

[0157] The above results, compared to that of Table 4, show that, atleast with low solubility gases, by lengthening the chain of thephospholipid fatty acid residues, one can dramatically increase thestability of the echogenic suspension toward pressure increases. Thiswas further confirmed by repeating the foregoing experiments butreplacing the phospholipid component by its higher homolog, i.e.,di-behenoylphosphatidylcholine (C₂₂ fatty acid residue). In this case,the resistance to collapse with pressure of the microbubbles suspensionswas still further increased.

[0158] Some of the microbubbles suspensions of this Example were testedin dogs as described previously for rabbits (imaging of the heartventricles after injection of 5 ml samples in the anterior cephalicvein). A significant enhancement of the useful in vivo echogenicresponse was noted, in comparison with the behavior of the preparationsdisclosed in Example 15, i.e., the increase in chain length of thefatty-acid residue in the phospholipid component increases the usefullife of the echogenic agent in vivo.

[0159] In the next Table below, there is shown the relative stability inthe left ventricle of the rabbit of microbubbles (SF₆) prepared fromsuspensions of a series of phospholipids whose fatty acid residues havedifferent chain lengths (<injected dose: 1 ml/rabbit). TABLE 6B Chainlength PC increment Δ Duration of Phospholipid (C_(n)) (Torr) P (Torr)contrast (sec) DMPC 14  57  37  31 DPPC 16 100  76  105 DSPC 18 115  95 120 DAPC 20 266 190 >300

[0160] It has been mentioned hereinabove that for the measurement ofresistance to pressure described in these Examples, a constant rate ofpressure rise of 100 Torr/min was maintained. This is justified by theresults given below which show the variations of the PC values fordifferent gases in function to the rate of pressure increase. In thesesamples DMPC was the phospholipid used. TABLE 7 PC (Torr) Gas Rate ofpressure increase (Torr/min) Sample 40 100 200 SF₆ 51 57 82 Air 39 50 62CH₄ 47 61 69 Xe 38 43 51 Freon 22 37 54 67

EXAMPLE 18

[0161] A series of albumin microballoons as suspensions in water wereprepared under air in a controlled sphere size fashion using thedirections given in Example 12. Then the air in some of the samples wasreplaced by other gases by the gas-exchange sweep method at ambientpressure. Then, after diluting to 1:10 with distilled water as usual,the samples were subjected to pressure testing as in Example 12. Fromthe results gathered in Table 7 below, it can be seen that the two-stepspreparation mode gives, in some cases, echo-generating agents withbetter resistance to pressure than the one-step preparation mode ofExample 12. TABLE 7 Sample Type of Mw of the Solubility Initial BubblePC No gas gas in water Count (10⁸/ml) (Torr) A14 Air 29 .0167 3.1 53 A18Air 29 .0167 3.8 52 A18/SF₆ SF₆ 146  .005 0.8 115  A18/C₂H₆ C₂H₆ 30 .0423.4 72 A19 Air 29 .0167 1.9 51 A19/SF₆ SF₆ 146  .005 0.6 140  A19/Xe Xe131  .108 1.3 67 A22/CF₄ CF₄ 88 .0038 1.0 167  A22/Kr Kr 84 .059 0.6 85

EXAMPLE 19

[0162] The method of the present invention was applied to an experimentas disclosed in the prior art, for instance Example 1 WO-92/11873. Threegrams of Pluronic® F 68 (a copolymer of polyoxyethylene-polyoxypropylenewith a molecular weight of 8400), 1 g of dipalmitoylphosphatidylglycerol(Na salt, Avanti Polar Lipids) and 3.6 g of glycerol were added to 80 mlof distilled water. After heating at about 80° C., a clear homogenoussolution was obtained. The tenside solution was cooled to roomtemperature and the volume was adjusted to 100 ml. In some experiments(see Table 8) dipalmitoylphosphatidylglycerol was replaced by a mixtureof diarachidoylphosphatldylcholine (920 mg) and 80 mg ofdipalmitoylphosphatidic acid (Na salt, Avanti Polar lipids).

[0163] The bubble suspensions were obtained by using two syringesconnected via a three-way valve. One of the syringes was filled with 5ml of the tenside solution while the other was filled with 0.5 ml of airor gas. The three-way valve was filled with the tenside solution beforeit was connected to the gas-containing syringe. By alternativelyoperating the two pistons, the tenside solutions were transferred backand forth between the two syringes (5 times in each direction), milkysuspensions were formed. After dilution (1:10 to 1:50) with distilledwater saturated at equilibrium with air, the resistance to pressure ofthe preparations was determined according to Example 12, the pressureincrease rate was 240 Torr/min. The following results were obtained:TABLE 8 Phospholipid Gas Pc (mm Hg) DP (mm Hg) DPPG air  28  17 DPPG SF₆138 134 DAPC/DPPA 9/1 air  46  30 DAPC/DPPA 9/1 SF₆ 269 253

[0164] It follows that by using the method of the invention andreplacing air with other gases, e.g., SF₆, even with known preparationsa considerable improvements, i.e., increase in the resistance topressure, may be achieved. This is true both in the case of negativelycharged phospholipids (e.g., DPPG) and in the case of mixtures ofneutral and negatively charged phospholipids (DAPC/DPPA).

[0165] The above experiment further demonstrates that the recognizedproblem sensitivity of microbubbles and microballoons to collapse whenexposed to pressure, i.e., when suspensions are injected into livingbeings, has advantageously been solved by the method of the invention.Suspensions with microbubbles or microballoons with greater resistanceagainst collapse and greater stability can advantageously be producedproviding suspensions with better reproducibility and improved safety ofechographic measurements performed in vivo on a human or animal body.

EXAMPLE 20

[0166] Multilamellar vesicles (MLVs) were prepared by dissolving 120 mgof diarachidoylphosphatidylcholine (DAPC, from Avanti Polar Lipids) and5 mg of dipalmitoylphosphatidic acid (DPPA acid form, from Avanti PolarLipids) in 25 ml of hexane/ethanol (8/2, v/v) then evaporating thesolvents to dryness in a round-bottomed flask using a rotary evaporator.The residual lipid film was dried in a vacuum desiccator and afteraddition of water (5 ml), the mixture was incubated at 90° C. for 30minutes under agitation. The resulting solution was extruded at 85° C.through a 0.8 μm polycarbonate filter (Nuclepore®). This preparation wasadded to 45 ml of a 167 mg/ml solution of dextran 10,000 MW (Fluka) inwater. The solution was thoroughly mixed, transferred in a 500 mlround-bottom flask, frozen at −45° C. and lyophilised under 13.33 Nt/m²(0.1 Torr). Complete sublimation of the ice was obtained overnight.Aliquots (100 mg) of the resulting lyophilisate were introduced in 20 mlglass vials. The vials were closed with rubber stoppers and the airremoved from vials using vacuum. Mixtures of air with various amounts ofsulfur hexafluoride were introduced into the vials via a needle throughthe stopper.

[0167] Bubble suspensions were obtained by injecting in each vial 10 mlof a 3% glycerol solution in water followed by vigorous mixing. Theresulting microbubble suspensions were counted using a hemacytometer.The mean bubble size was 2.0 μm. In vitro measurements (as defined inEP- A-0 554 213) of the critical pressure (Pc), echogenicity (i.e.,backscatter coefficient) and the bubble count for various samples wereperformed (see Table 9). TABLE 9 air SF₆ Echogenicity % % Q PC 1/(cm.sr)× Concentration Sample vol vol coeff. mmHg 100 (bubbles/ml) A 100   01.0  43 1.6 1.5 × 10⁸ B 95  5 1.3  68 2.1 1.4 × 10⁸ C 90 10 1.6  85 2.41.5 × 10⁸ D 75 25 3.1 101 2.3 1.4 × 10⁸ E 65 35 4.7 106 2.4 1.5 × 10⁸ F59 41 5.8 108 2.4 1.6 × 10⁸ G  0 100  722.3 115 2.3 1.5 × 10⁸

[0168] As it may be seen from the results, the microbubbles containing100% air (sample A) have a low resistance to pressure. However, withonly 5% SF₆, the resistance to pressure increases considerably (sampleB). With 25% SF₆ the resistance to pressure is almost identical to thatof 100% SF₆. On the other hand, the bubble concentrations, the meanbubble sizes and the backscatter coefficients are almost independent ofthe percentage of SF₆.

[0169] The resulting suspensions were injected intravenously intominipigs (Pitman Moore) at a dose of 0.5 ml per 10 kg and the images ofthe left ventricular cavity were recorded on a video recorder. In vivoechographic measurements were performed using an Acuson XP128 ultrasoundsystem (Acuson Corp. USA) and a 7 MHz sector transducer. The intensityof the contrast was measured by video densitometry using an imageanalyzer (Dextra Inc.). FIG. 2 shows the video densitometric recordingsin the left heart of a minipig. Again a considerable difference isobserved between the 100% air case (sample A) and the 95% air case(sample B). In particular, with 5% SF₆ the maximum intensity is alreadyalmost achieved and the half life in circulation shows also a very rapidincrease. With 10% SF₆, there is no additional increase in intensity butonly a prolongation of the half-life. From the example, it follows thatusing more than 10% to 25% SF₆ in the gas mixture provides no realbenefit. It is interesting to note that the values of the Q coefficientobtained for the mixtures used were well below the critical value of 5stipulated by WO-A-93/05819.

EXAMPLE 21

[0170] Aliquots (25 mg) of the PEG/DAPC/DPPA lyophilisate obtained asdescribed in Example 20 (using PEG 4000 instead of dextran 10,000) wereintroduced in 10 ml glass vials. Tedlar® sampling bags were filled withair and octafluorocyclobutane (C₄F₈). Known volumes were withdrawn fromthe bags by syringes and the contents thereof were mixed via a three waystopcock system. Selected gas mixtures were then introduced into theglass vials (previously evacuated). The lyophilisates were thensuspended in 2.5 ml saline (0.9% NaCl). The results presented below showthe resistance to pressure, the bubble concentration and the backscattercoefficient of the suspensions. In the case of 100% C₄F₈ the resistanceto pressure reached to 225 mm Hg (compared to 43 mm Hg in the case ofair). Again a considerable increase in pressure resistance was alreadyobserved with only 5% C₄F₈ (Pc=117 mmHg).

[0171] After intra-aortic injection in rabbits (0.03 ml/kg), a slightprolongation of the contrast effect in the myocardium was noticedalready with 2% C₄F₈ (when compared to air). However with 5% C₄F₈, theduration of the contrast increased considerably as if above a thresholdvalue in the resistance to pressure, the persistence of the bubblesincreases tremendously (see FIG. 3). TABLE 10 air C₄F₈ Echogenicity % %Q PC 1/(cm.sr) × Concentration Sample vol vol coeff. mmHg 100(bubbles/ml) A 100   0 1.0  43 1.6 1.8 × 10⁸ B 95  5 1.4 117 2.2 3.1 ×10⁸ C 90 10 1.7 152 3.1 4.7 × 10⁸ D 75 25 3.3 197 3.5 4.9 × 10⁸ E 65 354.6 209 3.4 4.3 × 10⁸ F 59 41 5.5 218 2.8 4.0 × 10⁸ G  0 100  1531 2252.3 3.8 × 10⁸

[0172] Here again, this combination of gases provided very good imagesat 5% of gas B in the mixture, while excellent images of the left heartwere obtained with the mixtures containing up to 25% ofoctafluorocyclobutane. Corresponding diagram of critical pressure as afunction of C₄F₈ in the mixture with air is given in FIG. 4. Thisexample again shows that the use of mixture of gases allows to improveconsiderably the resistance to pressure of air bubbles simply by addinga small percentage of a high molecular weight/low solubility gas. Thefigure further shows that by appropriate selection of the gas mixture itbecomes possible to obtain any desired resistance to pressure.

We claim:
 1. A method of ultrasound imaging comprising: administering toa subject a contrast agent comprising stabilized microbubbles, saidstabilized microbubbles comprising a physiologically acceptable gasselected from the group consisting of freons, halogenated hydrocarbons,and fluorinated gases, said stabilized microbubbles being stabilized atleast in part by a surfactant; and ultrasonically imaging said subject.2. A method of ultrasound imaging comprising: administering to a subjecta contrast agent comprising stabilized microbubbles, said stabilizedmicrobubbles comprising a physiologically acceptable gas that is afreon, said stabilized microbubbles being stabilized at least in part bya surfactant; and ultrasonically imaging said subject.
 3. The method ofclaim 1 wherein said stabilized microbubbles are suspended in an aqueousliquid carrier.
 4. The method of claim 1 wherein said stabilizedmicrobubbles are suspended in an aqueous liquid carrier which includes aviscosity enhancer.
 5. The method of claim 1 wherein the stabilizedmicrobubbles are between 0.5 and 10 microns in size.
 6. The method ofclaim 1 wherein the physiologically acceptable freon is selected fromthe group consisting of CF₄, CBrF₃, C₄F₈, CClF₃, C₂F₆, C₃F₈, C₄F₈,C₂ClF₃, CBRrClF₂, C₂Cl₂F₄, C₅F₁₀, C₅F₁₂, and C₄F₁₀.
 7. The method ofclaim 1 wherein the physiologically acceptable fluorinated gas isselected from the group consisting of SF₆, SeF₆, CF₄, CBrF₃, C₄F₈,CClF₃, C₂F₆, C₃F₈, C₄F₆, C₂ClF₅, CBrClF₂, C₂Cl₂F₄, C₅F₁₀, C₅F₁₂, andC₄F₁₀.
 8. The method of claim 2 wherein the physiologically acceptablefreon is selected from the group consisting of CF₄, CBrF₃, C₄F₈, CClF₃,C₂F₆, C₃F₈, C₄F₆, C₂ClF₅, CBrClF₂, C₂Cl₂F₄, C₅F₁₀, C₅F₁₂, and C₄F₁₀. 9.The method of claim 2 wherein the physiologically acceptable freon isselected from the group consisting of CF₄, C₄F₈, C₂F₆, C₃F₈, C₄F₆,C₅F₁₀, C₅F₁₂, and C₄F₁₀.
 10. The method of claim 1 wherein thephysiologicallky acceptable freon is selected from the group consistingof CF₄, C₂F₆, C₃F₈, C₄F₆, C₅F₁₀, C₅F₁₂, C₄F₈, and C₄F₁₀.
 11. The methodof claim 1 wherein the physiologically accepted fluorinated gascomprises SF₆.
 12. The method of claim 1 wherein the physiologicallyaccepted fluorinated gas comprises SeF₆.
 13. The method of claim 1wherein the physiologically accepted freon comprises CF₄.
 14. The methodof claim 1 wherein the physiologically accepted freon comprises CBrF₃.15. The method of claim 1 wherein the physiologically accepted freoncomprises C₄F₈.
 16. The method of claim 1 wherein the physiologicallyaccepted freon comprises CClF₃.
 17. The method of claim 1 wherein thephysiologically accepted freon comprises C₂F₆.
 18. The method of claim 1wherein the physiologically accepted freon comprises C₂ClF₅.
 19. Themethod of claim 1 wherein the physiologically accepted freon comprisesCBrClF₂.
 20. The method of claim 1 wherein the physiologically acceptedfreon comprises C₂Cl₂F₄.
 21. The method of claim 1 wherein thephysiologically accepted freon comprises C₄F₁₀.
 22. (Amended) The methodof claim 1 wherein the physiologically acceptable freon comprises C₃F₈.23. The method of claim 1 wherein the physiologically acceptable freoncomprises C₄F₆.
 24. The method of claim 1 wherein the physiologicallyacceptable freon comprises C₅F₁₀.
 25. The method of claim 1 wherein thephysiologically acceptable freon comprises C₅F₁₂.